The high-fat, low-carbohydrate ketogenic diet is a profoundly effective treatment for epileptic patients that reduces or eliminates seizure-activity while preserving normal neural function. Although this diet has been used to treat epilepsy for nearly a century, the mechanisms underlying this treatment are unclear. Because this treatment is very difficult for patients to follow, alternative therapies based on the mechanism of the diet are highly desirable. It is generally believed that ketone bodies, abundant in the blood of dieting patients, replace glucose as the primary substrate for brain metabolism, and that this metabolic shift may play a role in seizure protection. To better understand how the ketogenic diet confers seizure protection, it is important to establish a basic picture of how energy metabolism and activity of individual brain cells responds to changes in fuel supplies in vivo. Recently developed genetically-encoded fluorescent biosensors that report the ATP/ADP ratio and the NADH/NAD+ ratio provide the ability to perform live imaging of energy metabolism in individual brain cells. ATP and NADH are important parameters that play unique roles in cellular energy metabolism and are also hypothesized to have specific roles in the mechanism underlying the ketogenic diet. The ratio of cytosolic ATP/ADP reflects the energy status of the cell, and ketone body metabolism may lower ATP levels and activate ATP-sensitive potassium channels that reduce neuronal excitability. The cytosolic NADH/NAD+ ratio is primarily determined by the rate of glycolysis, the first steps in glucose metabolism. Cytosolic NADH may play an important role in long-term seizure protection, through its signaling to nuclear transcription factors. Understanding how ATP and NADH are influenced by neuronal activity and ketone body metabolism in live cells directly bears on these two proposals of the cellular mechanism for the ketogenic diet. This research proposal combines the use of ATP and NADH biosensors with advanced imaging techniques and electrical stimulation to examine energy dynamics in live neurons and astrocytes of the intact mammalian brain slice. The goal of Aim 1 is to understand how normal (physiological) and seizure-like (pathological) neuronal activity alters the global ATP and NADH levels in both astrocytes and neurons. This work will also examine whether glycolytic metabolism is engaged in response to activity-dependent energy depletion in both cell types. The goal of Aim 2 is to learn how ketone bodies alter the metabolic responses to neuronal activity. These studies will help identify whether ketone body metabolism produces a cellular state that is consistent with the hypothesized roles of ATP and NADH in the mechanisms of the ketogenic diet. Uncovering the cellular mechanisms of the ketogenic diet is an important step toward the goal of designing alternative treatments that are easier for patients to follow. PUBLIC HEALTH RELEVANCE: Approximately one third of children with epilepsy do not respond to drug treatment, and recurrent seizures can cause developmental delay or result in permanent disorder. Epilepsy has been effectively treated by a high- fat, very-low-carbohydrate diet for nearly a century, but the mechanism of this treatment is unknown. Understanding the relationship between metabolism and electrical activity at the level of individual brain cells is critical for uncovering the mechanisms of this treatment, and is a first step for designing alternative treatments that are less difficult for patients to follow.